Method of reacting an insolubilized enzyme in a fluid medium

ABSTRACT

Facilitating action of a particulate catalyst such as an insolubilized enzyme in a fluid medium by conducting the catalytic action while the particulate catalyst is in foraminous containers attached to a rotatable and/or reciprocatable stirrer shaft.

1Jnited States Patent Havewala et a1.

1 Oct. 23, 1973 METHOD OF REACTING AN INSOLUBILIZED ENZYME IN A FLUIDMEDIUM Inventors: Noshir B. l-lavewala, Corning;

Howard H. Weetall, Elmira, both of Assignee: Corning Glass Works,Corning,

Filed: June 14, 1971 Appl. No.: 152,507

US. Cl. 195/116, 23/288 R, 195/143,

252/477, 259/108, 195/63 Int. Cl C12b l/00 Field of Search 259/8, 23,43, 44,

References Cited UNITED STATES PATENTS Chisholm 23/288 E Harter 23/288 EWestmann.. 195/68 Miller 195/68 Primary ExaminerAlvin E. TanenholtzAssistant Examiner-William A. Simons Att0rneyClarence R. Patty, Jr. eta1.

ABSTRACT Facilitating action of a particulate catalyst such as aninsolubilized enzyme in a fluid medium by conducting the catalyticaction while the particulate catalyst is in foraminous containersattached to a rotatable and/or reciprocatable stirrer shaft.

2 Claims, 4 Drawing Figures PATENTEU OBI 2 3 I973 sum 1 or 2 TO POWERSOURCE INVENTORS. Nash/r B. Havewa/a Howard H. We era/l ATTORNEYPAIENIEllucr 23 um SHEET 2 [IF 2 IN VEN TORS. Nash/r B. Havewa/a HowardH. Weefal/ ATTORNEY METHOD OF REACTING AN INSOLUBILIZED ENZYME IN AFLUID MEDIUM BACKGROUND OF THE INVENTION FIELD OF THE INVENTION Thisinvention relates generally to a stirring apparatus and morespecifically to a stirring apparatus for use with a particulatecatalyst.

A catalyst is a substance that accelerates a chemical reaction andenables it to proceed under milder conditions than otherwise possible.Typically, catalysts are not affected by the reactions they promote.Catalysts may be either soluble or insoluble in a fluid reaction whichis catalyzed. In using insoluble catalysts, it is usually desirable toprovide a catalyst with a larger surface area per unit weight. For thisreason, many insoluble catalysts are used in a particulate and/or porousform.

Also, many otherwise soluble catalysts can be made insoluble byattachment to a water insoluble substance called a carrier. For example,in US. Pat. No. 3,519,538 there is taught a method for making normallysoluble enzymes insoluble by chemically coupling them to awater-insoluble carrier in such away that the enzymes do not lose theircatalytic power. There are many advantages associated withinsolubilizing otherwise soluble catalysts such as enzymes. For example,insolubilized catalysts can be easily removed from a reaction by readilyavailable means such as filtration or centrifugation. Thus, a catalyticreaction may be conveniently allowed to proceed for a measurable time.Further, since the insolubilized catalysts can be easily removed, andused repeatedly, the costs associated with use of more expensivecatalysts are greatly reduced.

As in the case of normally insoluble catalysts, it is usually desirableto use artificially insoluble catalysts in such a way that they offer alarge surface area per unit weight of catalyst. For this reason, thewater-insoluble carriers chosen for insolubilizing otherwise solublecatalysts are usually in a particulate and/or porous form. The insolublecarriers may be organic such as cellulose or any of various polymersavailable, or inorganic such as small glass beads or particles of porousglass.

In many respects, porous glass particles provide an ideal carrier forsuch catalysts as enzymes. For example, porous glass is dimensionallystable and it is relatively inert. Also, it can be easily cleaned orsterilized prior to catalyst attachment. Further, being porous, itoffers an extremely large surface area perunit weight (e.g., carriers ofpowdered porous 96 percent silica glass of 350Ai50A. pores of less than350 mesh are commonly used for insolubilizing enzymes). Thus, a greateramount of catalyst can be attached to the carrier by utilizing the innersurface area of the pores. By utilizing a porous carrier in comminutedor particulate form, an even greater carrier surface area is provided.Porous catalyst carriers or supports are being used more and moreextensively because of the large surface area per unit weight theyprovide.

However, for almost all solid catalyzed fluid phase reactions, porediffusion resistance can play an important role in determining the rateof reaction. Therefore, it has been found highly advantageous to utilizeporous catalyst supports in very small size particles, thereby reducingthe pore length through which reactants must pass to effectively utilizeavailable surface area to which the catalyst is attached.

Such particulate, porous catalyst carriers can be used in a variety ofchemical reactors. For example, porous catalyst supports may be used ina batch reactor, a continuous stirred tank reactor (CSTR), a fixed bedreactor, and a fluidized bed reactor.

In using the above reactors, however, it has been found the much desiredsmall catalyst support particle size has several disadvantages in itspractical applications. Industrial scale utilization of a particulatecatalyst support in a fixed bed reactor is, in many cases, impracticaldue to extremely high resistance to the fluid flow offered by that typeof packing. On the other hand, utilizing a particulate catalyst supportin a batch or CSTR reaction, frequently results in attrition of thecatalyst particles. The same problems are commonly encountered withfluidized bed reactors. There are yet other problems associated withusing particulate catalyst supports in the above reactors. For example,in using catalyst supports or carriers of high surface area there iscommonly encountered a film diffusion resistance on the particle surfacewhich hinders catalytic action. Also, in many catalytic reactions, solidproducts are formed which settle on the catalyst thereby graduallydiminishing available catalytic area. In addition, this makes itdifficult to replace or regenerate the catalyst composite, and, in somecases, to recover the product sought. Lastly, in many catalyticreactions, it is desirable to keep solids separate from the liquidphase, thereby limiting catalytic action to the liquid phase alone. Forexample, if reactants containing solid materials are utilized in a fixedbed reactor containing a catalyst support, the solid materials tend toclog the reactors. Thus, in view of the numerous disadvantagesassociated with the use of particulate and/or porous catalyst supports,there has been a long felt need for either a method or apparatus tofacilitate the use of such catalyst supports. The present inventionserves that need.

SUMMARY OF THE INVENTION We have surprisingly found that thedisadvantages associated with using particulate and/or porous catalystsupports can be overcome with a stirring device that can be used inbatch and continuous stirred tank reactors. The device consists of astirrer shaft that can be driven by conventional means such as by arotating and/or reciprocating motor or by hand, and one or moreforaminous-containers for the particulate catalyst which are attached tothe shaft. The foraminous containers also act as impellers when theshaft is rotatably and/or reciprocably driven in a fluid medium. Inpreferred embodiments, the containers comprise screen packets forholding the particulate catalyst while permitting inward and outwarddiffusion of reactants and products. The containers may be detachablymounted on the stirrer shaft and/or have closeable openings tofacilitate the replacement or regeneration of the particulate catalyst.By controlling the speed of the shaft in a fluid medium, film diffusionresistance can be easily controlled or at least minimized.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a side elevation view ofa stirrer embodying the principles of the present invention whereinthree of four screen packets for the particulate catalyst are shownradially attached to the stirrer shaft which is driven by a power sourcenot shown.

FIG. 2 is a top view of the stirrer shown in FIG. 1.

FIG. 3 is a bottom view of the stirrer shown in FIG.

FIG. 4 is a side elevation (partially exploded) of one of the containersshown in FIG. 1 which is detached from the stirrer shaft.

SPECIFIC EMBODIMENTS The stirrer of the present invention can be madefrom any materials which will not significantly affect the reaction inwhich they are used. The materials need only be sturdy enough to bedriven or agitated in a fluid medium without significant loss ofstructural integrity for the rotation and/or reciprocating speed chosenfor use in a medium of given viscosity. For example, if high speedrotation or reciprocation was anticipated in a relatively viscousreaction medium, it would be best to use materials of known structuralstability under adverse conditions, e.g., stainless steel can be used toform the shaft as well as the packets attached to the shaft by means ofstainless steel bolts. On the other hand, if a relatively slow agitatingspeed is used in a medium of low viscosity, the materials used toconstruct the stirrer could be of any lesser strength material whichwould not interfere with the reaction, e.g., plastics, glass, wood, andthe like. Of course, the materials comprising the shaft and parts of thepackets need not be the same.

As to the packets, the only requirements are that they be attachable tothe shaft in such a way that they function not only as containers forthe particulate catalyst, but also as impellers for the stirring device.As used herein, to function as an impeller means that the packets, whenattached to the shaft which is rotated, reciprocated, or otherwise movedabout in a fluid medium, tend to disrupt the apparent stillness of thefluid. The shaft can have one or more packets thus attached. In apreferred embodiment, more than one packet is attached to the shaft tobalance the rotation or other movement of the stirrer-shaft in use.Also, it is desirable to use more than one packet to carry greateramounts of the particulate catalyst and thus hasten the reaction, or, insome cases, to carry more than one catalyst.

The stirring assembly may be of any size commensurate with the magnitudeof the reaction desired. Thus, the stirrer may be only a few inches ingreatest diameter for use in a small beaker, or it may be many feetacross for use in a large tank.

The power source for driving the stirrer may be any conventional sourcesince it is not intended that the power source be a part of the presentinvention. Thus, for example, the shaft can be driven by rotation,reciprocation, eccentric rotation, or a combination of those means by alarge or small motor or can be manually driven by a hand crank once thestirrer is held in place by support means. In a preferred embodiment,the stirrer shaft is rotated by means of a controlled RPM mo- Theforaminous container for holding the particulate catalyst should havegenerally evenly distributed openings, small enough to adequatelycontain the particulate catalyst yet large enough to permit diffusion ofthe reactants into the container and thereby permit intimate contactwith the catalyst. The maximum size of the openings should be smallerthan the average particulate catalyst size. Likewise, the products ofcatalysis should be able to diffuse out of the container and into thereaction medium. The openings may be in the form of perforationsdistributed about the container or they may result from using a screenof known mesh size to construct a container on a relatively ridgedsupport structure. Stainless steel screen has been found to be anexcellent material from which to construct the containers. Thus, whenparticulate catalysts such as enzymes insolubilized by bonding to porousglass particles are used, a screening of 400 to 40 mesh NBS has beenfound suitable since the carrier particles commonly used will generallynot pass through a 40 mesh screen. It has been found that generally theparticulate catalyst used in the present invention should be at leastmesh size to minimize or eliminate the problems referred to above.

To understand the preferred embodiment shown in the drawings, a detaileddescription of the figures is given below.

FIG. 1 shows a side elevational view of a stirrer employing theprinciples of the present invention. In FIG. 1, there can be seen thatthe stirrer 5 consists of a cylindrical shaft 7 about which isfrictionally attached a flanged disc member 8. The flanged disc member 8may also be secured about the shaft 7 by means of one or more set screws(not shown) which pass through the collar portion 9 to engage the shaft7. The flanged portion 11 of the flanged disc member 8 is securelypositioned on the shaft 7 distal to the power source not shown. Radiallyattached to the disc portion 11 of the flanged disc member 8 arebox-like foraminous packets 13, three of which are shown. The relativeposition of a fourth foraminous packet can be seen in FIGS. 2 and 3discussed below. As can be generally seen, each of the foraminouspackets 13 shown in FIG. 1 have a boxlike shape having for two sidesmeshed screen members 21 held in place against a frame member (not shownin FIG. 1) by retaining members 15 secured to the frame members by fourscrews 19 on each screened side of the container.

FIG. 2 is a top view of the stirring device of FIG. 1 as seen from theplane indicated by lines 22 of FIG. 1. In FIG. 2 the flanged disc member3 engaged on the shaft 7 is shown in transparent form to show attachmentof four foraminous packets 13 more clearly. As can be seen from FIG. 2,the four packets 13 are radially attached about the shaft 7 by means ofscrews 23 which pass through openings in the disc portion 11 of theflanged disc member 8. After passing through the disc portion memberopenings, the screws 23 engage threaded openings (not shown in FIG. 2)in the top portions 25 of each packet 13. It can be seen that the screws23 securing the packets 13 to the disc portion 11 are positionedchordally with respect to the disc member 11 so as to secure the packetsabout the shaft 7 as closely as possible. Thus, the packets 13 are shownattached to the disc portion 11 in a staggered manner with the resultthat the attaching screws 23 in each packet engage two threaded openings(not shown) in the top portion 25 of each packet which are not in a lineparallel with any one side of the box-like packets 13.

FIG. 3 shows a bottom view of the stirrer of FIG. 1 as seen from theplane of lines 33 of FIG. 1. There, it can also be seen that the bottomportions 27 of the packets 13 occupy staggered positions about the shaft7 which may extend slightly through the center of the disc portion 11 ofthe flanged disc member.

FIG. 4 shows a partially exploded side elevational view of one of theforaminous packets 13 shown in FIGS. 1, 2, and 3 which is shown detachedfrom the flanged disc on the stirrer shaft. As can be seen, the frame 24of the packet 13 is a generally U-shaped member attachable to the discportion (not shown) at the top portion 25 of the shown U-shaped member24 by means of threaded openings 26 capable of engaging screws (notshown) which pass through similar openings in the disc portion to engagethe frame member. One screen retaining member 15 is shown in explodedform to show its attachment to the frame member by means of screws 19which pass through openings 20 to engage threaded openings 22 in theframe member 24. Also shown in exploded form is a removable end plate 17which can be held in place against the frame member 24 to form a closedcontainer by means of end plate screws 36 which pass through openings 35and 33 in the upper and lower end portions of the U-shaped frame member24. The end plate screws engage threaded openings 29 on ridge portions30 of the end plate 17 as shown in FIGS. 2 and 3, but not shown in FIG.4. The removable end plate 17 facilitates loading and unloading of theparticulate catalyst into or out of the foraminous packet 13. The endplate 17 also serves to hold those edges of the screen 21 not heldfirmly against the frame 24 by the retaining members 15 against theproximal inner portions of the retaining members as shown in FIG. 4.Thus, when the end plate 17 is secured in place by the end plateretaining screws 36 the screened packet is a closed foraminous containercapable of retaining the particulate catalyst while allowing diffusionof the reactants and products through the screen portions 21 of thepackets.

When the packets are attached to the stirrer shaft by means of theflanged disc member, they act as impellers when the shaft is rotated orreciprocally driven in a fluid medium. By attaching the packets as closeto the shaft as possible, as shown by the staggered arrangement of FIGS.1, 2, and 3, the structural integrity of the stirrer is assured evenunder high speed rotation and/or reciprocation in a relatively viscousreaction medium. In the specific stirrer just described, all materialscomprising the structure were stainless steel. The screen was made ofstainless steel and had a mesh size of 200 NBS. The screws were alsostainless steel.

It is, of course, not essential that the frame member 24 be U-shaped.For example, the frame may be made of several pieces which can bereadily bolted or screwed together. Also, the packets used not need bebox-like. They need only be attachable to the stirrer shaft (directly orindirectly) in such a manner that they act as impellers and asparticulate catalyst containers. Further, the packets may consist offoraminous or screen walls on all sides or any one or more sides of thepacket. Thus, for example, the packets may consist entirely of screeningin any three dimensional shape which will permit catalysis and animpeller-effect when the packets are attached to the shaft which isrotated in a fluid medium.

The particular stirrer described above through the figures is shown inapproximately the actual size of a stirrer which was used in a 2500 ml.flow-through beaker into which was continuously fed appropriatereactants for an enzymatic reaction. At the out-flow portion of thebeaker, enzymatic products were collected. These products were thecatalytic result of a particulate insolubilized enzyme on the reactantswhich flowed into the beaker. The out-flowing product concentration wasremarkably high and it is thought this was due to the novel design ofthe stirrer containing the particulate catalyst. In the above work, thestirrer was driven by a Bodine 1/70 HP Motor, 58 RPM, Type NSI-I 12R,single reduction type. The stainless steel stirrer shaft rotated by themotor was about 7 /z inches long and the motor was suitably mounted on asupport means above the beaker in such a way that the attached stirrerextended into the beaker to a point about 1 to 2 inches above thebeakers inner bottom. The stirrer was successfully used for continuousperiods of more than 4 weeks without adverse effect on the stirrer.

It will be apparent to those skilled in the art of catalysis that thereare many obvious modifications that can be made to the stirrer of thepresent invention to suit particular reaction needs. For example, thestirrer may have only one packet attached, or several. The packets maycontain one type of particulate catalyst, or several different catalystsin different packets. In certain enzymatic reactions, thesimultaneoususe of several catalysts is particularly desirable as is thesimultaneous use of one or more enzymes and one or more otherparticulate compounds which must be available for catalytic reaction.Lastly, the packets need not be used only for particulate catalysts thatremain essentially insoluble. For example, slowly dissolving particulatecatalysts can be used beneficially in the present invention to permit atime capsule release of the catalyst into the reaction medium.

Thus, it is intended that because of the many modifications possiblewith the present invention, the invention should be limited only by theappended claims.

We claim:

1. A method of using an insolubilized enzyme for an enzymatic reactionin fluid medium, the insolubilized enzyme comprising an enzymechemically coupled to a porous, particulate water-insoluble carriermaterial,

rous glass particles.

2. The method of claim 1 wherein the carrier to which the enzyme ischemically coupled consists of porous glass particles.